RESUMO
Calreticulin (Calr) is an endoplasmic reticulum (ER) chaperone involved in protein quality control, Ca2+ regulation and other cellular processes. The structure of Calr is unusual, reflecting different functions of the protein: a proline-rich ß-hairpin arm and an acidic C-terminal tail protrude from a globular core, composed of a ß-sheet sandwich and an α-helix. The arm and tail interact in the presence of Ca2+ and cover the upper ß-sheet, where a carbohydrate-binding site gives the chaperone glycoprotein affinity. At the edge of the carbohydrate-binding site is a conserved, strained disulphide bridge, formed between C106 and C137 of human Calr, which lies in a polypeptide-binding site. The lower ß-sheet has several conserved residues, comprised of a characteristic triad, D166-H170-D187, Tyr172 and the free C163. In addition to its role in the ER, Calr translocates to the cell surface upon stress and functions as an immune surveillance marker. In some myeloproliferative neoplasms, the acidic Ca2+-binding C-terminal tail is transformed into a polybasic sequence.
Assuntos
Calreticulina , Retículo Endoplasmático , Sítios de Ligação/genética , Calreticulina/genética , Calreticulina/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Transtornos MieloproliferativosRESUMO
The immune system homeostasis relies on a tight equilibrium of interconnected stimulatory and inhibitory signals. Disruption of this balance is characteristic of autoimmune diseases such as systemic lupus erythematosus (SLE). Aside from activating the classical complement pathway and enhancing pathogens and apoptotic cells phagocytosis, C1q has been recently shown to play an important role in immune modulation and tolerance by interacting with several inhibitory and stimulatory immune receptors. Due to its functional organization into collagen-like (CLR) and globular (GR) regions and its multimeric nature, C1q is able to interact simultaneously with several of these receptors and locally congregate pro- and anti-inflammatory signals, thus modulating the immune response. Leukocyte associated immunoglobulin-like (Ig-like) receptor 1 (LAIR-1), a ubiquitous collagen receptor expressed in many immune cell types, has been reported to interact with the CLR of C1q. In this study, we provide new insights into the molecular and structural determinants underlying C1q/LAIR-1 interaction. Recombinant LAIR-1 extracellular Ig-like domain was produced and tested for its interaction with C1q. A molecular dissection of C1q combined with competition assays reveals that LAIR-1 interacts with C1q's CLR through a binding site close but different from the one of its associated C1r2s2 proteases tetramer. On the other side, we identified LAIR-1 residues involved in C1q interaction by site-directed mutational analysis. All together, these results lead to propose a possible model for C1q interaction with LAIR-1 and will contribute to the fundamental understanding of C1q-mediated immune tolerance.
Assuntos
Colágeno/metabolismo , Complemento C1q/metabolismo , Receptores Imunológicos/metabolismo , Sítios de Ligação , Complemento C1q/genética , Humanos , Tolerância Imunológica , Mutação , Ligação Proteica , Receptores Imunológicos/genéticaRESUMO
C1 inhibitor (C1Inh) deficiency is responsible for hereditary angioedema (C1-INH-HAE) and caused by variants of the SERPING1/C1INH/C1NH gene. C1Inh is the major control of kallikrein-kinin system. C1Inh deficiency leads to its uncontrolled activation, with subsequent generation of the vasoactive peptide bradykinin. This update documents 748 different SERPING1 variants, including published variants and additional 120 unpublished ones. They were identified as heterozygous variants (n = 729), as homozygous variants in 10 probands and as compound heterozygous variants (nine combinations). Six probands with heterozygous variants exhibited gonadal mosaicism. Probands with heterozygous (n = 72) and homozygous (n = 1) variants were identified as de novo cases. Overall, 58 variants were found at positions showing high residue conservation among serpins, and have been referred to as a mousetrap function of C1Inh: reactive center loop, gate, shutter, breach, and hinge. C1Inh phenotype analysis identified dysfunctional serpin variants with failed serpin-protease association and a residual 105-kDa species after incubation with target protease. Regarding this characteristic, in conditions with low antigenic C1Inh, 74 C1-INH-HAE probands presented with an additional so-called intermediate C1-INH-HAE phenotype. The present update addresses a comprehensive SERPING1 variant spectrum that facilitates genotype-phenotype correlations, highlighting residues of strategic importance for serpin function and for identification of C1Inh deficiency as serpinopathy.
Assuntos
Angioedemas Hereditários/diagnóstico , Angioedemas Hereditários/genética , Proteína Inibidora do Complemento C1/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação , Fenótipo , Alelos , Proteína Inibidora do Complemento C1/química , Biologia Computacional , Bases de Dados Genéticas , Estudos de Associação Genética/métodos , Genótipo , Haploinsuficiência , Humanos , Modelos Moleculares , Conformação Proteica , Splicing de RNA , Relação Estrutura-AtividadeRESUMO
Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal-dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, joint hypermobility, and mild skin findings. A locus was mapped to an approximately 5.8 Mb region at 12p13.1 but no candidate gene was identified. In an international consortium we recruited 19 independent families comprising 107 individuals with pEDS to identify the locus, characterize the clinical details in those with defined genetic causes, and try to understand the physiological basis of the condition. In 17 of these families, we identified heterozygous missense or in-frame insertion/deletion mutations in C1R (15 families) or C1S (2 families), contiguous genes in the mapped locus that encode subunits C1r and C1s of the first component of the classical complement pathway. These two proteins form a heterotetramer that then combines with six C1q subunits. Pathogenic variants involve the subunit interfaces or inter-domain hinges of C1r and C1s and are associated with intracellular retention and mild endoplasmic reticulum enlargement. Clinical features of affected individuals in these families include rapidly progressing periodontitis with onset in the teens or childhood, a previously unrecognized lack of attached gingiva, pretibial hyperpigmentation, skin and vascular fragility, easy bruising, and variable musculoskeletal symptoms. Our findings open a connection between the inflammatory classical complement pathway and connective tissue homeostasis.
Assuntos
Complemento C1r/genética , Complemento C1s/genética , Síndrome de Ehlers-Danlos/genética , Deleção de Genes , Mutação de Sentido Incorreto , Periodontite/genética , Adolescente , Adulto , Criança , Pré-Escolar , Mapeamento Cromossômico , Cromossomos Humanos Par 12/genética , Síndrome de Ehlers-Danlos/diagnóstico , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Exoma , Feminino , Loci Gênicos , Humanos , Masculino , Linhagem , Periodontite/diagnóstico , Conformação Proteica , Adulto JovemRESUMO
Nanodiamonds are promising nanomedicines for diagnostic and therapeutic applications. As nanodiamonds are mainly administered intravenously, it is critical to understand the humoral immune response upon exposure to nanodiamonds. Here, we report the interactions of pristine, oxidized, and PEG-functionalized nanodiamonds with human complement, an important part of our humoral innate immunity. In particular, we report the nanodiamond binding properties of the recognition protein of the classical complement pathway: C1q, which also takes part in many other physiological and pathological processes. Our results show similar trends in the effects of C1q on the three types of nanodiamonds. Complement activation assays using human serum show that the nanodiamonds trigger slight activities via the alternative pathway and no response via the classical pathway. Nevertheless, surface plasmon resonance shows that C1q binds the nanodiamonds and transmission electron microscopy reveals their agglutination. Studies with macrophages further show that C1q attachment affects their phagocytosis and cytokine response.
Assuntos
Ativação do Complemento , Complemento C1q/metabolismo , Imunidade Inata , Nanodiamantes/química , Aglutinação , Difusão Dinâmica da Luz , Humanos , Macrófagos/metabolismo , Nanodiamantes/ultraestrutura , Células THP-1 , TermogravimetriaRESUMO
Bacterial pathogens recruit circulating proteins to their own surfaces, co-opting the host protein functions as a mechanism of virulence. Particular attention has focused on the binding of plasminogen (Plg) to bacterial surfaces, as it has been shown that this interaction contributes to bacterial adhesion to host cells, invasion of host tissues, and evasion of the immune system. Several bacterial proteins are known to serve as receptors for Plg including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytoplasmic enzyme that appears on the cell surface in this moonlighting role. Although Plg typically binds to these receptors via several lysine-binding domains, the specific interactions that occur have not been documented in all cases. However, identification of the relevant residues could help define strategies for mitigating the virulence of important human pathogens, such as Streptococcus pneumoniae (Sp). To shed light on this question, we have described a combination of peptide-spot array screening, competition and SPR assays, high-resolution crystallography, and mutational analyses to characterize the interaction between SpGAPDH and Plg. We identified three SpGAPDH lysine residues that were instrumental in defining the kinetic and thermodynamic parameters of the interaction. Altogether, the integration of the data presented in this work allows us to propose a structural model for the molecular interaction of the SpGAPDH-Plg complex.
Assuntos
Plasminogênio/metabolismo , Streptococcus pneumoniae/patogenicidade , Sequência de Aminoácidos , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Cinética , Ligação Proteica , Conformação Proteica , Ressonância de Plasmônio de Superfície , TermodinâmicaRESUMO
Human L-ficolin is a soluble protein of the innate immune system able to sense pathogens through its fibrinogen (FBG) recognition domains and to trigger activation of the lectin complement pathway through associated serine proteases. L-Ficolin has been previously shown to recognize pneumococcal clinical isolates, but its ligands and especially its molecular specificity remain to be identified. Using solid-phase binding assays, serum and recombinant L-ficolins were shown to interact with serotype 2 pneumococcal strain D39 and its unencapsulated R6 derivative. Incubation of both strains with serum triggered complement activation, as measured by C4b and C3b deposition, which was decreased by using ficolin-depleted serum. Recombinant L-ficolin and its FBG-like recognition domain bound to isolated pneumococcal cell wall extracts, whereas binding to cell walls depleted of teichoic acid (TA) was decreased. Both proteins were also shown to interact with two synthetic TA compounds, each comprising part structures of the complete lipoteichoic acid molecule with two PCho residues. Competition studies and direct interaction measurements by surface plasmon resonance identified PCho as a novel L-ficolin ligand. Structural analysis of complexes of the FBG domain of L-ficolin and PCho revealed that the phosphate moiety interacts with amino acids previously shown to define an acetyl binding site. Consequently, binding of L-ficolin to immobilized acetylated BSA was inhibited by PCho and synthetic TA. Binding of serum L-ficolin to immobilized synthetic TA and PCho-conjugated BSA triggered activation of the lectin complement pathway, thus further supporting the hypothesis of L-ficolin involvement in host antipneumococcal defense.
Assuntos
Lectinas/metabolismo , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/metabolismo , Ácidos Teicoicos/metabolismo , Acetilação , Parede Celular/metabolismo , Ativação do Complemento , Complemento C3b/metabolismo , Complemento C4b/metabolismo , Fibrinogênio/genética , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Lectinas/genética , Fosforilcolina/química , Ligação Proteica , Estrutura Terciária de Proteína/genética , Streptococcus pneumoniae/imunologia , Ressonância de Plasmônio de Superfície , Ácidos Teicoicos/química , FicolinasRESUMO
Complement receptor type 1 (CR1) is a membrane receptor expressed on a wide range of cells. It is involved in immune complex clearance, phagocytosis, and complement regulation. Its ectodomain is composed of 30 complement control protein (CCP) modules, organized into four long homologous repeats (A-D). In addition to its main ligands C3b and C4b, CR1 was reported to interact with C1q and mannan-binding lectin (MBL) likely through its C-terminal region (CCP22-30). To decipher the interaction of human CR1 with the recognition proteins of the lectin complement pathway, a recombinant fragment encompassing CCP22-30 was expressed in eukaryotic cells, and its interaction with human MBL and ficolins was investigated using surface plasmon resonance spectroscopy. MBL and L-ficolin were shown to interact with immobilized soluble CR1 and CR1 CCP22-30 with apparent dissociation constants in the nanomolar range, indicative of high affinity. The binding site for CR1 was located at or near the MBL-associated serine protease (MASP) binding site in the collagen stalks of MBL and L-ficolin, as shown by competition experiments with MASP-3. Accordingly, the mutation of an MBL conserved lysine residue essential for MASP binding (K55) abolished binding to soluble CR1 and CCP22-30. The CR1 binding site for MBL/ficolins was mapped to CCP24-25 of long homologous repeat D using deletion mutants. In conclusion, we show that ficolins are new CR1 ligands and propose that MBL/L-ficolin binding involves major ionic interactions between conserved lysine residues of their collagen stalks and surface exposed acidic residues located in CR1 CCP24 and/or CCP25.
Assuntos
Lectina de Ligação a Manose da Via do Complemento , Lectina de Ligação a Manose/metabolismo , Receptores de Complemento/metabolismo , Sítios de Ligação , Proteínas de Transporte/metabolismo , Lectina de Ligação a Manose da Via do Complemento/genética , Humanos , Cinética , Lectinas/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Receptores de Complemento/química , Receptores de Complemento/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , FicolinasRESUMO
Background: Hereditary angioedema (HAE) is a potentially life-threatening disorder characterized by recurrent episodes of subcutaneous or submucosal swelling. HAE with normal C1 inhibitor (HAE-nC1-INH) is an underdiagnosed condition. Although the association with genetic variants has been identified for some families, the genetic causes in many patients with HAE-nC1-INH remain unknown. The role of genes associated with bradykinin catabolism is not fully understood. Objective: We sought to investigate the biological parameters and the genes related to kallikrein-kinin system in families with a clinical phenotype of HAE-nC1-INH and presenting with a carboxypeptidase N (CPN) deficiency. Methods: This study includes 4 families presenting with HAE-nC1-INH and CPN deficiency. Patients' clinical records were examined, biological parameters of kallikrein-kinin system were measured, and genetics was analyzed by next-generation sequencing and Sanger sequencing. Predictive algorithms (Human Splicing Finder, Sorting Intolerant From Tolerant, Polymorphism Phenotyping v2, MutationTaster, and ClinPred) were used to classify variants as affecting splicing, as benign to deleterious, or as disease-causing. Results: Patients presented with angioedema and urticaria, mainly on face/lips, but also with abdominal pain or laryngeal symptoms. Affected patients displayed low CPN activity-30% to 50% of median value in plasma. We identified 3 variants of the CPN1 gene encoding the catalytic 55-kDa subunit of CPN: c.533G>A, c.582A>G, and c.734C>T. CPN deficiency associated with genetic variants segregated with HAE-nC1-INH symptoms in affected family members. Conclusions: CPN1 gene variants are associated with CPN deficiency and HAE-nC1-INH symptoms in 4 unrelated families. Genetic CPN deficiency may contribute to bradykinin and anaphylatoxin accumulation, with synergistic effects in angioedema and urticarial symptoms.
RESUMO
Complement activation is considered to contribute to the pathogenesis of severe SARS-CoV-2 infection, mainly by generating potent immune effector mechanisms including a strong inflammatory response. Involvement of the lectin complement pathway, a major actor of the innate immune anti-viral defense, has been reported previously. It is initiated by recognition of the viral surface Spike glycoprotein by mannose-binding lectin (MBL), which induces activation of the MBL-associated protease MASP-2 and triggers the proteolytic complement cascade. A role for the viral nucleoprotein (N) has also been reported, through binding to MASP-2, leading to protease overactivation and potentiation of the lectin pathway. In the present study, we reinvestigated the interactions of the SARS-CoV-2 N protein, produced either in bacteria or secreted by mammalian cells, with full-length MASP-2 or its catalytic domain, in either active or proenzyme form. We could not confirm the interaction of the N protein with the catalytic domain of MASP-2 but observed N protein binding to proenzyme MASP-2. We did not find a role of the N protein in MBL-mediated activation of the lectin pathway. Finally, we showed that incubation of the N protein with MASP-2 results in proteolysis of the viral protein, an observation that requires further investigation to understand a potential functional significance in infected patients.
Assuntos
COVID-19 , Lectina de Ligação a Manose da Via do Complemento , Serina Proteases Associadas a Proteína de Ligação a Manose , SARS-CoV-2 , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/imunologia , Humanos , SARS-CoV-2/imunologia , Lectina de Ligação a Manose da Via do Complemento/imunologia , COVID-19/imunologia , COVID-19/virologia , Ligação Proteica , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Ativação do Complemento/imunologia , Lectina de Ligação a Manose/metabolismo , Lectina de Ligação a Manose/imunologia , FosfoproteínasRESUMO
Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, lack of attached gingiva and thin and fragile gums leading to gingival recession. Connective tissue abnormalities of pEDS typically include easy bruising, pretibial plaques, distal joint hypermobility, hoarse voice, and less commonly manifestations such as organ or vessel rupture. pEDS is caused by heterozygous missense mutations in C1R and C1S genes of the classical complement C1 complex. Previously we showed that pEDS pathogenic variants trigger intracellular activation of C1r and/or C1s, leading to extracellular presence of activated C1s. However, the molecular link relating activated C1r and C1s proteases to the dysregulated connective tissue homeostasis in pEDS is unknown. Using cell- and molecular-biological assays, we identified activated C1s (aC1s) as an enzyme which degrades collagen I in cell culture and in in vitro assays. Matrix collagen turnover in cell culture was assessed using labelled hybridizing peptides, which revealed fast and comprehensive collagen protein remodeling in patient fibroblasts. Furthermore, collagen I was completely degraded by aC1s when assays were performed at 40°C, indicating that even moderate elevated temperature has a tremendous impact on collagen I integrity. This high turnover is expected to interfere with the formation of a stable ECM and result in tissues with loose compaction a hallmark of the EDS phenotype. Our results indicate that pathogenesis in pEDS is not solely mediated by activation of the complement cascade but by inadequate C1s-mediated degradation of matrix proteins, confirming pEDS as a primary connective tissue disorder.
Assuntos
Complemento C1s , Síndrome de Ehlers-Danlos , Humanos , Colágeno Tipo I/genética , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/patologia , Mutação de Sentido Incorreto , Complemento C1s/genéticaRESUMO
Complement C1s association with the pathogenesis of several diseases cannot be simply explained only by considering its main role in activating the classical complement pathway. This suggests that non-canonical functions are to be deciphered for this protease. Here the focus is on C1s cleavage of HMGB1 as an auxiliary target. HMGB1 is a chromatin non-histone nuclear protein, which exerts in fact multiple functions depending on its location and its post-translational modifications. In the extracellular compartment, HMGB1 can amplify immune and inflammatory responses to danger associated molecular patterns, in health and disease. Among possible regulatory mechanisms, proteolytic processing could be highly relevant for HMGB1 functional modulation. The unique properties of HMGB1 cleavage by C1s are analyzed in details. For example, C1s cannot cleave the HMGB1 A-box fragment, which has been described in the literature as an inhibitor/antagonist of HMGB1. By mass spectrometry, C1s cleavage was experimentally identified to occur after lysine on position 65, 128 and 172 in HMGB1. Compared to previously identified C1s cleavage sites, the ones identified here are uncommon, and their analysis suggests that local conformational changes are required before cleavage at certain positions. This is in line with the observation that HMGB1 cleavage by C1s is far slower when compared to human neutrophil elastase. Recombinant expression of cleavage fragments and site-directed mutagenesis were used to confirm these results and to explore how the output of C1s cleavage on HMGB1 is finely modulated by the molecular environment. Furthermore, knowing the antagonist effect of the isolated recombinant A-box subdomain in several pathophysiological contexts, we wondered if C1s cleavage could generate natural antagonist fragments. As a functional readout, IL-6 secretion following moderate LPS activation of RAW264.7 macrophage was investigated, using LPS alone or in complex with HMGB1 or some recombinant fragments. This study revealed that a N-terminal fragment released by C1s cleavage bears stronger antagonist properties as compared to the A-box, which was not expected. We discuss how this fragment could provide a potent brake for the inflammatory process, opening the way to dampen inflammation.
Assuntos
Complemento C1s , Proteína HMGB1 , Humanos , Complemento C4/metabolismo , Lipopolissacarídeos , Anti-InflamatóriosRESUMO
C1q, the recognition subunit of the C1 complex of complement, is an archetypal pattern recognition molecule with the striking ability to sense a wide variety of targets, including a number of altered self-motifs. The recognition properties of its globular domain were further deciphered by means of x-ray crystallography using deoxy-D-ribose and heparan sulfate as ligands. Highly specific recognition of deoxy-D-ribose, involving interactions with Arg C98, Arg C111, and Asn C113, was observed at 1.2 A resolution. Heparin-derived tetrasaccharide interacted more loosely through Lys C129, Tyr C155, and Trp C190. These data together with previous findings define a unique binding area exhibiting both polyanion and deoxy-D-ribose recognition properties, located on the inner face of C1q. DNA and heparin compete for C1q binding but are poor C1 activators compared with immune complexes. How the location of this binding area in C1q may regulate the level of C1 activation is discussed.
Assuntos
Complemento C1q/química , Desoxirribose/química , Heparitina Sulfato/química , Estrutura Terciária de Proteína , Sítios de Ligação , Complemento C1q/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Desoxirribose/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Ligação Proteica , Triptofano/química , Triptofano/metabolismo , Tirosina/química , Tirosina/metabolismoRESUMO
CUB domains are 110-residue protein motifs exhibiting a ß-sandwich fold and mediating protein-protein interactions in various extracellular proteins. Recent X-ray structural and mutagenesis studies have led to the identification of a particular CUB domain subset, cbCUB (Ca(2+)-binding CUB domain). Unlike other CUB domains, these harbour a homologous Ca(2+)-binding site that underlies a conserved binding site mediating ionic interaction between two of the three conserved acidic Ca(2+) ligands and a basic (lysine or arginine) residue of a protein ligand, similar to the interactions mediated by the low-density lipoprotein receptor family. cbCUB-mediated protein-ligand interactions usually involve multipoint attachment through several cbCUBs, resulting in high-affinity binding through avidity, despite the low affinity of individual interactions. The aim of the present review is to summarize our current knowledge about the structure and functions of cbCUBs, which represent the majority of the known CUB repertoire and are involved in a variety of major biological functions, including immunity and development, as well as in various cancer types. Examples discussed in the present review include a wide range of soluble and membrane-associated human proteins, as well as some archaeal and invertebrate proteins. The fact that these otherwise unrelated proteins share a common Ca(2+)-dependent ligand-binding ability suggests a mechanism inherited from very primitive ancestors. The information provided in the present review should stimulate further investigations on the crucial interactions mediated by cbCUB-containing proteins.
Assuntos
Motivos de Aminoácidos , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Sequência de Aminoácidos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Our immune system responds to infectious (PAMPs) and tissue damage (DAMPs) signals. The complement system and alarmin High-Mobility Group Box 1 (HMGB1) are two powerful soluble actors of human host defense and immune surveillance. These systems involve molecular cascades and amplification loops for their signaling or activation. Initially activated as alarm raising systems, their function can be finally switched towards inflammation resolution, where they sustain immune maturation and orchestrate repair mechanisms, opening the way back to homeostasis. However, when getting out of control, these defense systems can become deleterious and trigger serious cellular and tissue damage. Therefore, they can be considered as double-edged swords. The close interaction between the complement and HMGB1 pathways is described here, as well as their traditional and non-canonical roles, their functioning at different locations and their independent and collective impact in different systems both in health and disease. Starting from these systems and interplay at the molecular level (when elucidated), we then provide disease examples to better illustrate the signs and consequences of their roles and interaction, highlighting their importance and possible vicious circles in alarm raising and inflammation, both individually or in combination. Although this integrated view may open new therapeutic strategies, future challenges have to be faced because of the remaining unknowns regarding the molecular mechanisms underlying the fragile molecular balance which can drift towards disease or return to homeostasis, as briefly discussed at the end.
Assuntos
Proteína HMGB1 , Alarminas , Proteínas do Sistema Complemento , Proteína HMGB1/metabolismo , Humanos , Inflamação , Transdução de Sinais/fisiologiaRESUMO
C1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca(2+)-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340-19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB(1)-EGF-CUB(2) interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model.
Assuntos
Complemento C1/química , Complemento C1r/química , Complemento C1s/química , Espectrometria de Massas/métodos , Estrutura Quaternária de Proteína , Sequência de Aminoácidos , Sítios de Ligação , Ativação do Complemento , Complemento C1/genética , Complemento C1/metabolismo , Complemento C1r/genética , Complemento C1r/metabolismo , Complemento C1s/genética , Complemento C1s/metabolismo , Humanos , Lisina/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Coloração e Rotulagem/métodos , Propriedades de SuperfícieRESUMO
Ficolins are oligomeric innate immune recognition proteins consisting of a collagen-like region and a fibrinogen-like recognition domain that bind to pathogen- and apoptotic cell-associated molecular patterns. To investigate their carbohydrate binding specificities, serum-derived L-ficolin and recombinant H- and M-ficolins were fluorescently labeled, and their carbohydrate binding ability was analyzed by glycan array screening. L-ficolin preferentially recognized disulfated N-acetyllactosamine and tri- and tetrasaccharides containing terminal galactose or N-acetylglucosamine. Binding was sensitive to the position and orientation of the bond between N-acetyllactosamine and the adjacent carbohydrate. No significant binding of H-ficolin to any of the 377 glycans probed could be detected, providing further evidence for its poor lectin activity. M-ficolin bound preferentially to 9-O-acetylated 2-6-linked sialic acid derivatives and to various glycans containing sialic acid engaged in a 2-3 linkage. To further investigate the structural basis of sialic acid recognition by M-ficolin, point mutants were produced in which three residues of the fibrinogen domain were replaced by their counterparts in L-ficolin. Mutations G221F and A256V inhibited binding to the 9-O-acetylated sialic acid derivatives, whereas Y271F abolished interaction with all sialic acid-containing glycans. The crystal structure of the Y271F mutant fibrinogen domain was solved, showing that the mutation does not alter the structure of the ligand binding pocket. These analyses reveal novel ficolin ligands such as sulfated N-acetyllactosamine (L-ficolin) and gangliosides (M-ficolin) and provide precise insights into the sialic acid binding specificity of M-ficolin, emphasizing the essential role of Tyr(271) in this respect.
Assuntos
Carboidratos/química , Lectinas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Cristalografia por Raios X , Fibrinogênio/química , Fibrinogênio/genética , Humanos , Lectinas/química , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Ácido N-Acetilneuramínico/química , Polissacarídeos/química , Análise Serial de Proteínas , Ligação Proteica , Tirosina , FicolinasRESUMO
Ficolins are innate immune recognition proteins involved in activation of the lectin complement pathway. These oligomeric lectin-like proteins are assembled from subunits consisting of a collagen-like triple helix and a trimeric fibrinogen-like recognition domain. In humans, three ficolins coexist: they differ in their ligand binding specificities, but share the capacity to associate with proteases through their collagen-like stalks and trigger complement activation. We describe methods to decipher the recognition specificities of ficolins, based on surface plasmon resonance, an optical technique allowing real-time and label-free monitoring of biomolecular interactions. This technique was mainly used to characterize and compare binding of the three recombinant full-length ficolins and of their isolated recognition domains to various immobilized BSA-glycoconjugates, acetylated BSA or biotinylated heparin. The avidity phenomenon that enhances the apparent affinity of interactions between oligomeric lectin-like proteins and the multivalent ligands is also discussed.
Assuntos
Lectinas/química , Lectinas/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Animais , Sítios de Ligação , Células CHO , Células Cultivadas , Cricetulus , Drosophila , Humanos , Cinética , Lectinas/farmacologia , Ligantes , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Especificidade por Substrato , FicolinasRESUMO
Complement component C1q, a soluble defense collagen, is the recognition protein of the classical complement pathway. C1q is able to recognize and interact with multiple targets and, via the subsequent activation of its cognate serine proteases C1r and C1s, initiates the complement cascade. C1q is made up of six ABC heterotrimers each containing two different functional regions, an N-terminal collagen-like region (CLR) and a C-terminal globular region (GR). These heterotrimers assemble via their N-terminal regions, resulting in the characteristic 'bouquet-like' shape of C1q with an N-terminal bundle of collagen fibers with six diverging stems each exhibiting a C-terminal globular head. The GRs are responsible for the versatile recognition of multiple C1q targets, whereas the CLRs trigger immune response through interacting with several cellular or soluble partners. We report here the generation of the first recombinant form of human C1q without its recognition globular heads. The noncollagenous domain 2 (nc2) of type IX collagen has been substituted for the C1q GR in order to control the correct registering of the collagen triple helices of C1q chains A, B, and C. The resulting CLR_nc2 recombinant protein produced in stably transfected EXPI293 mammalian cells was correctly assembled and folded, as demonstrated by mass spectrometry, mass photometry, and electron microscopy experiments. Its interaction properties were investigated using surface plasmon resonance analysis with known CLR ligands: the tetramer of C1r and C1s dimers and MBL-associated protein MAp44. Comparison with the interaction properties of native serum-derived C1q and CLR revealed that recombinant CLR_nc2 retains C1q CLR functional properties.